@article{trihemasava_chakraborty_blackburn_xu_2020, title={Expression, purification, and phylogenetic analysis of MDIS1-INTERACTING RECEPTOR-LIKE KINASE1 (MIK1)}, volume={39}, ISSN={["1573-4943"]}, DOI={10.1007/s10930-020-09926-9}, abstractNote={An abundance of protein structures has been solved in the last six decades that are paramount in defining the function of such proteins. For unsolved protein structures, however, predictions based on sequence and phylogenetic similarity can be useful for identifying key domains of interaction. Here, we describe expression and purification of a recombinant plant LRR-RLK ectodomain MIK1 using a modified baculovirus-mediated expression system with subsequent N-linked glycosylation analysis using LC-MS/MS and computational sequence-based analyses. Though highly ubiquitous, glycosylation site specificity and the degree of glycosylation influenced by genetic and exogenous factors are still largely unknown. Our experimental analysis of N-glycans on MIK1 identified clusters of glycosylation that may explicate the regions involved in MIK1 ectodomain binding. Whether these glycans are necessary for function is yet to be determined. Phylogenetic comparison using multiple sequence alignment between MIK1 and other LRR-RLKs, namely TDR in Arabidopsis thaliana, revealed conserved structural motifs that are known to play functional roles in ligand and receptor binding.}, number={5}, journal={PROTEIN JOURNAL}, author={Trihemasava, Krittin and Chakraborty, Sayan and Blackburn, Kevin and Xu, Guozhou}, year={2020}, month={Oct}, pages={461–471} }
 @misc{chakraborty_nguyen_wasti_xu_2019, title={Plant Leucine-Rich Repeat Receptor Kinase (LRR-RK): Structure, Ligand Perception, and Activation Mechanism}, volume={24}, ISSN={["1420-3049"]}, DOI={10.3390/molecules24173081}, abstractNote={In recent years, secreted peptides have been recognized as essential mediators of intercellular communication which governs plant growth, development, environmental interactions, and other mediated biological responses, such as stem cell homeostasis, cell proliferation, wound healing, hormone sensation, immune defense, and symbiosis, among others. Many of the known secreted peptide ligand receptors belong to the leucine-rich repeat receptor kinase (LRR-RK) family of membrane integral receptors, which contain more than 200 members within Arabidopsis making it the largest family of plant receptor kinases (RKs). Genetic and biochemical studies have provided valuable data regarding peptide ligands and LRR-RKs, however, visualization of ligand/LRR-RK complex structures at the atomic level is vital to understand the functions of LRR-RKs and their mediated biological processes. The structures of many plant LRR-RK receptors in complex with corresponding ligands have been solved by X-ray crystallography, revealing new mechanisms of ligand-induced receptor kinase activation. In this review, we briefly elaborate the peptide ligands, and aim to detail the structures and mechanisms of LRR-RK activation as induced by secreted peptide ligands within plants.}, number={17}, journal={MOLECULES}, author={Chakraborty, Sayan and Nguyen, Brian and Wasti, Syed Danyal and Xu, Guozhou}, year={2019}, month={Sep} }
 @article{chakraborty_trihemasava_xu_2018, title={Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells}, ISSN={["1940-087X"]}, DOI={10.3791/58283}, abstractNote={It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.}, number={138}, journal={JOVE-JOURNAL OF VISUALIZED EXPERIMENTS}, author={Chakraborty, Sayan and Trihemasava, Krittin and Xu, Guozhou}, year={2018}, month={Aug} }
 @article{chakraborty_pan_tang_woolard_xu_2018, title={The Extracellular Domain of Pollen Receptor Kinase 3 is structurally similar to the SERK family of co-receptors}, volume={8}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-018-21218-y}, abstractNote={Abstract}, journal={SCIENTIFIC REPORTS}, author={Chakraborty, Sayan and Pan, Haiyun and Tang, Qingyu and Woolard, Colin and Xu, Guozhou}, year={2018}, month={Feb} }
 @article{li_chakraborty_xu_2017, title={Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions}, volume={12}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0175317}, abstractNote={Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Ǻ and 2.75 Ǻ respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).}, number={4}, journal={PLOS ONE}, author={Li, Zhijie and Chakraborty, Sayan and Xu, Guozhou}, year={2017}, month={Apr} }
 @article{li_chakraborty_xu_2016, title={X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa}, volume={72}, ISSN={["2053-230X"]}, DOI={10.1107/s2053230x16014278}, abstractNote={Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses inArabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein,Camelina sativalectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space groupC222 orC2221, with unit-cell parametersa= 94.7,b= 191.5,c= 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, author={Li, Zhijie and Chakraborty, Sayan and Xu, Guozhou}, year={2016}, month={Oct}, pages={782–787} }